Production of cellulolytic and xylanolytic enzymes by an Arthrographis species

A fungal isolate, Arthrographis sp. strain F4, when grown in shake-flask culture, produced cellulolytic and xylanolytic enzymes optimally at 30°C with an initial pH of 5.0 to 6.0. Coarsely-ground filter paper was the most suitable carbon substrate for production of the enzymes. Inorganic nitrogen sources gave higher activities of the enzymes than organic nitrogen sources: NH₄NO₃ and yeast extract was the most effective combination. Significant stimulation (P<0.05) of enzyme production was achieved with 0.1% (v/v) Tween 80.B.C. Okeke was and S.K.C. Obi is with the Department of Microbiology, University of Nigeria, Nsukka, Nigeria. B.C. Okeke is now with the Department of Bioscience and Biotechnology, Royal College Building, University of Strathclyde, Glasgow G1 1XW, UK     

Production of cellulolytic enzymes from theXylaria andHypoxylon species of xylariaceae

Eighteen strains of xylariaceous fungi have been screened for higher activities of cellulolytic enzymes,Trichoderma reesei QM 9414 was also examined for comparison. Strains ofXylaria anisopleura andX. regalis had higher endocellulase (CMCase) and exocellulase (Avicelase) activities after 2 weeks' incubation.Hypoxylon stygium produced the highest activity of -glucosidase 3 days after inoculation. The optimum pH for these cellulolytic enzymes was approx. 5.0 and the optimum temperatures ranged from 37 to 50°C. A mixed culture process usingT. reesei QM 9414 andH. stygium was developed to obtain enhanced synthesis of cellulase. -Glucosidase activities in the mixed culture increased within 48h whenH. stygium was introduced after 24h.     

Production of cellulolytic enzymes by immobilized Sporotrichum thermophile.

Spores of Sporotrichum thermophile were immobilized in agar, polyacrylamide, and sodium alginate to generate in situ mycelium for production of cellulolytic enzymes. Immobilized mycelium was considerably less effective than free cells for cellulase productivity. Of the three gel types, agar beads proved to be the best carrier for the immobilized spores and subsequently generated mycelium. Results of repeated batch experiments suggested that the immobilized mycelia could be reused but at much reduced efficiency.     

Production of chitinase by Fusarium species

The presence of chitinase activity without inducers in the enzymic precipitates from the culture fluid of 25-day-old autolyzed cultures of 17 Fusarium species has been studied. In all cases endochitinase and -N-acetylglucosaminidase activities were found. The chitinase activity as a joint action of these two enzymes with production of N-acetylglucosamine was also determined. A correlation among endochitinase, -N-acetylglucosaminidase, and chitinase was always found. Fusarium oxysporum f.sp. lini, F. subglutinans, and F. moniliforme were the best producers of chitinase activity. Fusarium species could be a good source of chitinases for production by fungi.     

Production of mycelial protein and cellulolytic enzymes from food wastes

Summary Extracted grape waster material and pressed apple pulp were tested as carbon sources forPenicillium funiculosum 515,Myrothecium verrucaria 9095 andAspergillus niger TMF-15. They were good growth substrates, especially forA. niger. When cultivated on mixed substrate in optimized nutrient medium,A. niger accumulated a product of 35% crude protein with a maximum productivity of 0.117 g protein/1/h and cellulose consumption of 90.92%.A. niger also produced the highest levels of cellulase activity. Maximum carboxymethyl cellulase and activity against filter paper were 494 units/l and 97 units/l, respectively.     

Production of mycelial protein and hydrolytic enzymes from paper mill sludges by cellulolytic fungi

Summary Characterization of lignocellulosic wastes from three paper mills in New York State indicated that a kraft mill sludge contained substantial quantities of utilizable cellulose and hemicellulose. This residue was tested as a carbon source for seven cellulolytic fungi.Trichoderma reesei DAOM 167654 accumulated a product of over 22% crude protein, and caused a conversion of sludge to protein of almost 15% in 3 days growth in shake flasks.T. reesei also produced the highest levels of cellulase, whileT. longibrachiatum produced more xylanase (35 units/ml) than other fungi examined.     

Production of cellulolytic enzymes containing cinnamic acid esterase from Schizophyllum commune

To develop enzyme preparations capable of digesting plant biomass, we examined the production of cinnamic acid esterase as well as cellulolytic and xylanolytic enzymes in cultures of Schizophyllum commune. The cinnamic acid esterase was produced in the cultures containing solid cellulosic substrates, with production being enhanced by delignifying the wood powder. This indicates that these esterases are produced by cellulose, despite their substrates being phenolic compounds. Cellulolytic and xylanolytic enzymes, with the exception of α-arabinofuranosidase, were also produced in cultures containing cellulosic substances. These results show that enzyme preparation can have high activity of cinnamic acid esterase and cellulolytic and xylanolytic enzymes when S. commune is incubated in the presence of cellulose. These enzyme preparations will be useful for digesting plant biomass and for releasing cinnamic acid derivatives from plant cell walls.     

The production of cellulolytic enzymes by fungal cultures

Summary Twelve fungal cultures belonging to the genera ofAspergillus, Tricboderma, Chaetomium, Stachybotrys, andHypocrea were screened for the production of cellulolytic activity. All twelve were found to degrade xylan, avicel, and carboxymethylcellulose, More cellulolytic activity was obtained with shaken cultures than with still cultures and the addition of citrate-phosphate buffer to the media greatly depressed the levels of cellulolytic activity. Varying the composition of the mineral salts in the medium had no effect on the cellulolytic activity.The growth ofAspergillus wentii under controlled conditions in a bioreactor showed that the cellulolytic activity was not affected by the aeration rate or the type of stirrer. The rate of stirring, however, did effect the cellulolytic activity, as at lower stirring speeds considerable wall growth occurred which resulted in low levels of cellulolytic activity.Culture supernatant fromAspergillus wentii was found to hydrolyze from 30–32% of Solka-Floc and from 2–10% of corn cobs, wheat straw, and newsprint. The extensive hydrolysis of the Solka-Floc indicates that with suitably treated cellulosic wastes and appropriate enzymes, appreciable amounts of sugars could be obtained.     

Production of multiple xylanolytic and cellulolytic enzymes by thermophilic fungus Myceliophthora sp. IMI 387099

This study reports the production of xylanolytic and cellulolytic enzymes by a thermophilic fungal isolate Myceliophthora sp. using a cheap medium containing rice straw and chemically defined basal medium under solid-state culture. A combination of one factor at a time approach followed by response surface methodology using Box-Behnken design of experiments resulted in 2.5, 1.25, 1.28 and 4.23 fold increase in xylanase, endoglucanase, beta-glucosidase and FPase activity, respectively. The zymograms developed against IEF gels showed that multiple isoforms of xylanase (5), endoglucanase (4) and beta-glucosidase (2) were produced under optimized culture conditions. Moreover, thiol containing serine proteases produced during the growth of the culture had no role in the post-translational modification of these xylanases.     

Production of ligninolytic enzymes by Fusarium solani strains isolated from different substrata

A comparative study on the extracellular ligninolytic enzymatic activity of five strains of Fusarium solani in a carbon-limited medium under shaking, revealed a differential production of these enzymes. Aryl alcohol oxidase (AAO) activity was observed only in the supernatant of strain CLPS no. 568 with levels higher than 57 mU ml⁻¹. Free extracellular laccase activity was detected in strains CLPS nos. 493, 568 and 570, strain no. 568 being the one which showed the highest activity (over 8.6 mU ml⁻¹). Free extracellular lignin peroxidase (LiP) activity was not detected in any isolate tested, whereas low levels of manganese-dependent peroxidase (MnP) and manganese-independent peroxidase (MIP) activities were detected in certain isolates used. The AAO activity of F. solani on primary α-alcohols such as veratryl alcohol, is reported for the first time; this enzyme activity is hydrogen-peroxide independent. This is also the first report for extracellular MnP and MIP activities of F. solani. This revised version was published online in November 2006 with corrections to the Cover Date.     

Production of protoplasts from Fusarium scirpi by lytic enzymes from Streptomyces tsusimaensis

Summary The ability of serveral strains of Streptomyces to degrade cell walls from Fusarium scirpi was tested by plating them on agar containing a cell wall preparation derived from the fungus. In this assay, S. tsusimaensis was most effective in producing a clear zone of lysis during growth on the opaque medium. This Streptomyce strain was subsequently grown in liquid culture containing cell walls as the sole carbon source and the exoenzymes were isolated from the culture broth. The enzyme preparation produces a clear zone of lysis when filled into wells in the cell wall agar and was used to prepare protoplasts from F. scirpi. The protoplast yield was 1x10⁹ protoplasts/ml of enzyme solution from 35 mg dry weight of Fusarium mycelium. Protoplasts could be regenerated at a frequency of up to 80%.     

Production of xylanolytic enzymes in association with the cellulolytic activities of Penicillium funiculosum

Penicillium funiculosum produced 16 and 0.4 units ml^?1 of d-xylanase (1,4-β-d-xylan xylanohydrolase, EC 3.2.1.8) and β-d-xylosidase (1,4-β-d-xylan xylohydrolase, EC 3.2.1.37), respectively, in shake flasks. Both enzymes were 100% stable when heated at 50°C for 30 min and on prolonged heating d-xylanase and β-d-xylosidase showed 46 and 20% loss, respectively. Maximum hydrolysis (75%) of d-xylan was obtained when the end products were removed. The addition of β-d-xylosidase markedly influenced the degree of hydrolysis of d-xylan. End-product analysis of the d-xylan hydrolysate showed the presence of d-xylose, d-xylobiose, d-xylotriose, d-xylotetraose, d-xylopentose and l-arabinose. The fractionation of culture filtrate of Penicillium funiculosum grown on cellulose powder or in a combination of cellulose powder and wheat bran indicated the presence of two d-xylanases. The role of cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] and d-xylanase on the overall hydrolysis of pure cellulose and lignocellulosic substrates is discussed.     

Production of yeast-lytic enzymes by Cytophaga species in batch culture

The conditions for culture storage, inoculum preparation, and growth of a Cytophaga species, constitutive with respect to yeast-lytic enzymes, have been established in shake-flask studies and in 5 liter fermentor experiments. A low cost medium was adopted for 900 liter-scale fermentation and gave an enzyme activity in the fermentation broth somewhat greater than the comparable laboratory-scale one.     

Purification and characterization of cellulolytic enzymes produced by Aspergillus nidulans

Three exo-glucanases, two endo-glucanases and two β-glucosidases were separated and purified from the culture medium of Aspergillus nidulans. The optimal assay conditions for all forms of cellulase components ranged from pH 5.0 to 6.0 and 50°C and 65°C for exo-glucanases and endo-glucanases but 35°C and 65°C for β-glucosidases. A close relation of enzyme stability to their optimal pH range was observed. All the cellulase components were stable for 10 min at 40–50°C. Exo-II and Exo-III ( K _m, 38.46 and 37.71 mg/ml) had greater affinity for the substrate than Exo-I ( K _m, 50.00 mg/ml). The K _m values of Endo-I and Endo-II (5.0 and 4.0 mg/ml) and their maximum reaction velocities ( V _max, 12.0 and 10.0 IU/mg protein) were comparable. β-Glucosidases exhibited K _m values of 0.24 and 0.12 mmol and V _max values of 8.00 and 0.67 IU/mg protein. The molecular weights recorded for various enzyme forms were: Exo-I, 29000; Exo-II, 72500; Exo-III, 138000; Endo-I, 25000; Endo-II, 32500; β-Gluco-I, 14000 and β-Gluco-II, 26000. Exo- and endo-glucanases were found to require some metal ions as co-factors for their catalytic activities whereas β-glucosidases did not. Hg²⁺ inhibited the activity of all the cellulase components. The saccharification studies demonstrated a high degree of synergism among all the three cellulase components for hydrolysis of dewaxed cotton.